Method of elevating photosynthesis speed of plant by improving pyruvate phosphate dikinase

ABSTRACT

The present invention relates to the transformation of C4 plants. It also relates to the high-level expression of foreign genes in C4 plants. More specifically, the present invention relates to the creation of C4 plants retaining an excellent photosynthetic capacity at low temperature by achieving high-level expression of an enzyme constituting the C4 photosynthetic pathway. In the present invention, C4 plants are transformed using an expression cassette that comprises a promoter, a C4 plant genomic gene, under control of said promoter, encoding an enzyme constituting a photosynthetic pathway, and a terminator. The C4 plant genomic gene encoding an enzyme constituting a photosynthetic pathway is preferably a C4 plant genome-derived PPDK gene or a modified form thereof. The present invention is particularly useful in improving the production of C4 plants having PPDK (especially, in improving the production of maize under low temperature conditions).

FIELD OF THE INVENTION

The present invention relates to the transformation of C4 plants. It also relates to the high-level expression of foreign genes in C4 plants. More specifically, the present invention relates to the creation of C4 plants retaining an excellent photosynthetic capacity at low temperature by achieving high-level expression of an enzyme constituting the C4 photosynthetic pathway. The present invention is particularly useful in improving the production of C4 plants having PPDK (especially, in improving the production of maize under low temperature conditions).

BACKGROUND OF THE INVENTION

Plant pyruvate, orthophosphate dikinase (PPDK; EC2.7.9.1) is one of the important enzymes constituting the C4 cycle and it has been believed that there is a high correlation between PPDK activity and photosynthesis rate in C4 plants. Further, PPDK has the lowest activity among enzymes constituting the C4 cycle; the PPDK reaction has been regarded as a rate-limiting stage of C4 photosynthesis. Also, PPDK is a tetramer composed of four subunits which are weakly associated with each other. When exposed to low temperature conditions (at or below 12° C.), PPDK is known to be dissociated into dimers or monomers, thus rapidly losing its activity. In general, maize PPDK will lose approximately 70% of its activity when treated at 0° C. for 20 minutes. Meanwhile, the activity of maize PPDK was measured at various temperatures to prepare an Arrhenius plot, indicating that there was an inflection point at 11.7° C., which was found to match the critical temperature for maize growth. In view of these points, a decrease in PPDK activity has been regarded as the main factor responsible for slowdown of photosynthesis in C4 plants at low temperature.

Flaveria brownii (F. brownii), an Asteraceae plant, is categorized as a C3/C4 intermediate type and its PPDK is known to be hardly deactivated even when treated at a temperature as low as 0° C. (Burnell J N, A comparative sturdy of the cold-sensitivity of pyruvate, Pi dikinese in Flaveria species, Plant Cell Physiol., 31:295-297 (1990)). Hence, it was expected that this cold-tolerant PPDK gene could be used to create C4 plants capable of C4 photosynthesis at a lower temperature, i.e., C4 plants more resistant to cold.

In the previous studies, the inventors of the present invention succeeded in determining a region important for cold tolerance of PPDK by isolation and DNA sequencing of the F. brownii PPDK gene. They also demonstrated that the sequence of this region could be used to convert cold-sensitive PPDK into a cold-tolerant form through recombination between this sequence and the DNA of cold-sensitive PPDK derived from other plant (WO95/15385; Usami S, Ohta S, Komari T, Burnell J N, Cold stability of pyruvate, orthophosphate dikinase of Flaveria brownii, Plant Mol Biol, 27:969-80 (1995); Ohta S, Usami S, Ueki J, Kumashiro T, Komari T, Burnell J N, Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate, orthophosphate dikinase, FEBS Lett, 396:152-6 (1996)).

Through many studies of maize transformants, however, the inventors of the present invention found that effects of the artificially introduced PPDK (introduced PPDK) were masked by naturally-occurring PPDK in C4 plants (endogenous PPDK). This would be attributed to an abundance of endogenous PPDK constituting several percent of soluble proteins in C4 plants. Further, the inventors of the present invention found that heterotetramers could be formed between introduced PPDK subunits and endogenous PPDK subunits.

To overcome the phenomenon where effects of the introduced PPDK are masked by the endogenous PPDK, two techniques are available, one of which involves increasing the expression level of the introduced PPDK and the other is inhibition of the endogenous PPDK. The former involves integrating a sequence (e.g., intron) into a gene construct for transformation (in most cases, an intron(s) being integrated between a promoter and a structural gene) to increase the expression level of an externally introduced gene (WO96/30510: PLD intron, WO97/47755: double-ligated introns). The latter is an antisense technique for inhibiting the expression level of an object gene by introduction of a gene whose mRNA has a sequence complementary to mRNA from the object gene to be inhibited from expression (Japanese Patent No. 2651442 and its divisional patent No. 2694924). The inventors of the present invention have tried these techniques.

In increasing the expression level of the introduced PPDK by integration of an intron(s) etc., a gene construct used for transformation was constructed in a general manner through ligation between a maize PPDK promoter (Glackin Calif., Grula J W, Organ-specific transcripts of different size and abundance derive from the same pyruvate, orthophosphate dikinase gene in maize, PNAS, 87:3004-3008 (1990)) and a cDNA molecule of the PPDK gene derived from F. brownii, F. bidentis (Usami S, Ohta S, Komari T, Burnell J N, Cold stability of pyruvate, orthophosphate dikinase of Flaveria brownii, Plant Mol Biol, 27:969-80 (1995)) or maize (Matsuoka M, Structure, genetic mapping, and expression of the gene for pyruvate, orthophosphate dikinase from maize, J. Biol. Chem, 265:16772-16777 (1990)). At the same time, the inventors of the present invention attempted to increase the expression level of the introduced PPDK by inserting any one of the following introns between the promoter and the structural gene: Intron 1 of the Castor bean catalase gene (Ohta S, Mita S, Hattori T, Nakamura K, Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence, Plant Cell Physiol, 31:805-813 (1990)), Intron 1 of the rice phospholipase D gene (Ueki J, Morioka S, Komari T, Kumashiro T, Purification and characterization of phospholipase D from rice and maize (Zea mays L.), Plant Cell Physiol, 36:903-914 (1995)), Intron 1 of maize ubiquitin (Christensen et al., (1992)) and Intron 1 of maize Shrunken-1 (Vasil V, Clancy M, Ferl R J, Vasil I K, Hannah L C, Increased gene expression by the first intron of maize Shrunken-1 locus in grass species, Plant Physiol, 91:1575-1579 (1989)). Further, based upon a report suggesting that repeated introns resulted in an increased expression level (Ueki J, Ohta S, Morioka S, Komari T, Kuwata S, Kubo T, Imaseki H, The synergistic effects of two-intron insertions on heterologous gene expression and advantages of the first intron of a rice gene for phospholipase D, Plant Cell Physiol, 40:618-623 (1999)), the inventors of the present invention also attempted to insert multiple repeated introns.

DISCLOSURE OF THE INVENTION

The studies of the present inventors indicated that these introns resulted in an increased expression level in the order: Intron 1 of Shrunken-1<Intron 1 of the Castor bean catalase gene<Intron 1 of the rice phospholipase D gene and that these introns, when inserted in combination, resulted in a greater increase in expression level than when inserted alone, for example, Intron 1 of the rice phospholipase D gene resulted in an increased expression level when combined with Intron 1 of the Castor bean catalase gene or Intron 1 of maize ubiquitin.

However, even the most improved transformants expressed the introduced PPDK at a level as low as around 700 μg/g of fresh green leaves, which was only about half that of the endogenous PPDK; there was no remarkable effect resulting from the introduced PPDK. Although the expression level of the introduced PPDK is regarded as high compared to other artificially introduced genes, it would be impossible to clarify effects of the introduced gene in a case where there are abundant products of the endogenous gene (i.e., where the endogenous gene is highly expressed) unless the endogenous gene is inhibited.

In contrast, an antisense gene for inhibition of the endogenous PPDK was constructed based on a 395 bp sequence of the maize PPDK gene covering from SacI in the 5′-untranslated region to EcoRI in Intron 1, 6 repeated copies of this sequence, or a cDNA-derived PstI fragment (2.4 kb) covering almost all segments of the maize PPDK mature enzyme. The constructed antisense gene was used to transform maize. The reason why attention was directed to the 395 bp sequence was because this sequence corresponded to a transit peptide segment, for which low homology was shared between maize PPDK and F. brownii PPDK, and hence it would be able to selectively inhibit the maize PPDK alone.

As a consequence, there was no inhibitory effect on expression levels was observed in simple introduction of the antisense gene for the 395 bp sequence. With low frequency, some transformants modified to inhibit their endogenous PPDK appeared in a case where maize was transformed with an antisense gene for the 6 repeated copies of the 395 bp sequence or the cDNA-derived PstI fragment (2.4 kb). However, such transformants modified to inhibit their endogenous PPDK also had a reduced level of the introduced PPDK and therefore did not achieve specific inhibition of the endogenous PPDK alone. Further, such transformants were too weak to grow and most of them withered and died before maturation because inhibition occurred on PPDK essential for C4 plants, including both the endogenous PPDK and the introduced PPDK. Also, no seed production was seen in even those transformants that came into flower. It was theoretically impossible to selectively control the expression of genes sharing a very similar sequence by the antisense technique in view of its mechanism, thus indicating that such a technique could not be adapted in this case.

There is an attempt to introduce a genomic gene instead of cDNA to achieve high-level expression, in C3 plants, of a gene for an enzyme constituting the C4 photosynthetic pathway (JP 10-248419 A). In this attempt, however, a gene that is not present in C3 plants (or, if any, expressed at a very low level) is merely introduced from C4 plants. It would be much more difficult to achieve further expression, in C4 plants, of genes naturally occurring in C4 plants or high-level expression, in C4 plants, of genes encoding proteins which are already expressed in abundance in C4 plants, like photosynthesis-related enzymes, than to achieve expression of C4 plants-derived genes in C3 plants.

A variety of previous biochemical studies have estimated that PPDK might be a rate-limiting factor of C4 photosynthesis. However, no conclusive evidence has been established that these previous studies were correct because there was no report showing actual high-level expression of PPDK in C4 plants or artificial introduction of PPDK with new properties.

As stated above, the cDNA-based technique could not increase expression of the introduced gene to a sufficient level even in combination with an intron(s) etc., while the antisense technique was unable to selectively inhibit the endogenous gene alone. For this reason, the inventors of the present invention tried to introduce a genomic gene.

Meanwhile, the studies of the present inventors indicated that when a cold-tolerant F. brownii PPDK cDNA was introduced as such into maize, the introduced gene was expressed at a very low level. This would be because F. brownii is a C3/C4 intermediate plant and inherently produces a smaller amount of PPDK than C4 plants. Further, the inventors of the present invention have found that expression of F. brownii PPDK in C4 plants was improved by constructing a chimeric gene between a pure C4 plant F. bidentis or maize and F. brownii.

In turn, the inventors of the present invention made a variety of studies on genomic gene introduction based on the maize PPDK genomic gene, instead of the genomic gene for C3/C4 intermediate F. brownii PPDK. As a result, they found that the expression level of PPDK in maize was increased by simple introduction of the maize PPDK genomic gene into maize and that the expression level of maize PPDK under cold cultivation was increased using a gene that was modified into a cold-tolerant type by mutagenesis in a certain region of the maize PPDK genomic gene. These findings led to the completion of the present invention.

The present invention provides a method for increasing the expression level of an enzyme constituting a photosynthetic pathway in a C4 plant, comprising transforming the C4 plant using an expression cassette that comprises a promoter, a C4 plant genomic gene, under control of said promoter, encoding the enzyme, and a terminator. It also provides a transgenic C4 plant obtainable by the method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows procedures for construction of #2706 in Example 1.

FIG. 2A shows the amount of PPDK present in progeny plants of transformants modified with a maize genomic gene (#2706), compared between individuals homozygous or heterozygous for the introduced PPDK gene (homo or hetero) and individuals null for the gene (null). The vertical axis represents the expression level (μg/gfwt) of PPDK contained per g of fresh green leaves, and each bar represents standard error. There was a significant difference between homo or hetero individuals and null individuals at a significance level of 1%.

FIG. 2B shows the photosynthesis rate at various leaf surface temperatures in progeny plants of transformants modified with a maize genomic gene (#2706), compared between homo or hetero individuals and null individuals. The vertical axis represents the photosynthesis rate (μmol CO₂·m⁻²·s⁻¹), and each bar represents standard error. At a leaf surface temperature of 8° C., there was a significant difference between homo or hetero individuals and null individuals at a significance level of 5%.

FIG. 3 shows procedures for introduction of F. brownii-type mutations into the PPDK genome.

FIG. 4 shows procedures for construction of #2838 in Example 2.

FIG. 5A shows the maize PPDK amino acid sequence (SEQ ID NO: 17) FIG. 5B shows the F. brownii PPDK amino acid sequence (SEQ ID NO: 18). The line-encircled amino acid sequence is an approximately 1/6 region from the C-terminal, which is important for cold tolerance.

FIG. 5C shows a comparison of the C-terminal 1/6 region between maize PPDK (residues 824-947 of SEQ ID NO: 17) and F. brownii PPDK (residues 833-955 of SEQ ID NO: 18) amino acid sequences. The line-encircled amino acid residues are different between them.

FIG. 6A shows the amount of PPDK present in progeny plants of transformants modified with a mutated maize genomic gene (#2838), compared between homo or hetero individuals and null individuals. The vertical axis represents the expression level (μg/gfwt) of PPDK contained per g of fresh green leaves, and each bar represents standard error.

FIG. 6B shows the photosynthesis rate at various leaf surface temperatures in progeny plants of transformants modified with #2838, compared between homo or hetero individuals and null individuals. The vertical axis represents the photosynthesis rate (μmol CO₂·m⁻²·s⁻¹), and each bar represents standard error.

FIG. 7A shows the amount of PPDK present at various leaf surface temperatures in progeny plants of transformants modified with a mutated maize genomic gene (#2838), compared between homo or hetero individuals and null individuals. The vertical axis represents the expression level (μg/gfwt) of PPDK contained per g of fresh green leaves, and each bar represents standard error.

FIG. 7B shows the photosynthesis rate at various leaf surface temperatures in progeny plants of transformants modified with #2838, compared between homo or hetero individuals and null individuals. The vertical axis represents the photosynthesis rate (μmol CO₂·m⁻²·s⁻¹), and each bar represents standard error. At leaf surface temperatures of 20° C. and 13° C., there was a significant difference between homo or hetero individuals and null individuals at a significance level of 5%. At a leaf surface temperature of 8° C., there was a significant difference at a significance level of 1%.

FIG. 8 shows a cold inactivation pattern of PPDK in transformants with a mutated maize genomic gene. The horizontal axis represents time on ice (min), while the vertical axis represents PPDK activity (%). Open triangle (Δ), open square (□), solid triangle (▴), solid circle (●) and open circle (◯) represents transformants with PPDK contents of 4152.3 μg/gfwt, 9122.4 μg/gfwt, 1390.6 μg/gfwt, 9802.8 μg/gfwt and 1292.1 μg/gfwt, respectively, relative to 1 g of desalted fresh green leaves. Cross (×) represents F. brownii and plus (+) represents the maize inbred line A188 (1495.2 μg/gfwt). Transformants with high PPDK contents could retain their PPDK activity, even on ice, at almost the same level as F. brownii.

DETAILED DESCRIPTION OF THE INVENTION

Various types of enzymes are known to be included in the “enzyme constituting the C4 photosynthetic pathway” as used herein. Among these enzymes, the method of the present invention can be used to increase the expression levels of enzymes that are expressed at relatively low levels, easily deactivated, and/or involved in the rate-limiting stage of the photosynthetic pathway. In particular, the method of the present invention can be used to increase the expression levels of enzymes that have the properties as mentioned above under low temperature conditions (e.g., at or below about 12° C.). PPDK is known as an example of such enzymes. Also, the method of the present invention can be used to enhance the photosynthetic capacity at low temperature in C4 plants having a limit for growth under low temperature conditions (e.g., at or below about 12° C.).

In a case where the method of the present invention is used to create transgenic C4 plants, a gene consisting of any one of the following DNA molecules may be used as a “C4 plant genomic gene encoding an enzyme constituting a photosynthetic pathway.” The method using such a gene is particularly preferred to obtain maize plants modified to enhance their photosynthesis rate under low temperature conditions:

(a) the maize PPDK genomic gene, i.e., a DNA molecule consisting of the nucleotide sequence of Nos. 1732-8508 of SEQ ID NO: 15;

(b) a DNA molecule consisting of a nucleotide sequence derived from the nucleotide sequence of Nos. 1732-8508 of SEQ ID NO: 15 by deletion, substitution, addition or insertion of one or more nucleotides, and encoding a protein possessing PPDK activity;

(c) a DNA molecule being hybridizable under stringent conditions to the DNA molecule being complementary to the DNA molecule consisting of the nucleotide sequence of Nos. 1732-8508 of SEQ ID NO: 15, and encoding a protein possessing PPDK activity; and

(d) a DNA molecule consisting of a nucleotide sequence being at least 50% (preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90%, particularly preferably at least 95%, and most preferably at least 98%) homologous to the nucleotide sequence of Nos. 1732-8508 of SEQ ID NO: 15, and encoding a protein possessing PPDK activity. To calculate a homology between nucleotide sequences, a commercially available software package may be used. As used herein, the term “stringent conditions” refers to hybridization conditions of a temperature of at least about 40° C., a salt concentration of about 6×SSC (1×SSC=15 mM sodium citrate buffer; pH 7.0; 0.15 M sodium chloride) and 0.1% SDS, preferably at least about 50° C., more preferably at least about 65° C.

Alternatively, a gene encoding any one of the following proteins may be used:

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 17;

(b) a protein consisting of an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 17 by deletion, substitution, addition or insertion of one or more amino acids, and possessing PPDK activity;

(c) PPDK derived from a C4 plant; and

(d) a protein consisting of an amino acid sequence being at least 50% (preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90%, particularly preferably at least 95%, and most preferably at least 98%) homologous to the amino acid sequence of SEQ ID NO: 17, and possessing PPDK activity. The term “homology” when used herein for amino acid sequences generally means that the extent of the similarity between amino acid residues constituting the respective sequences to be compared. In this sense, the presence of gaps and the nature of amino acids are taken into account. To calculate a homology, a commercially available software package may be used.

As used herein, the term “genomic gene” encompasses a genome-derived gene per se and a modified form thereof, unless otherwise specified. Also as used herein, the term “C4 plant genomic gene encoding an enzyme constituting a photosynthetic pathway” encompasses a C4 plant genome-derived gene per se and a modified form thereof. Such a modified gene is preferably modified to have one or more nucleotide substitutions such that the amino acid sequence of the enzyme encoded by the gene is equivalent to the amino acid sequence of the corresponding enzyme in plants with desired characteristics (e.g., cold tolerance), preferably in C4 plants or plants of an intermediate nature between C3 and C4, more preferably in plants belonging to Flaveria, even more preferably in F. brownii or F. bidentis. Preferably, such a substitution is allowed to occur exclusively in exon segments, with as many intron segments as possible being retained intact. The number of bases to be substituted is not particularly limited, but it is preferably about 1 to 50, more preferably about 1 to 40, and most preferably about 1 to 30 (e.g., 29) when the enzyme constituting a photosynthetic pathway is PPDK. Likewise, the number of amino acids to be substituted is preferably about 1 to 40, more preferably about 1 to 30, and most preferably about 1 to 20 (e.g., 17 as shown in FIG. 5) when the enzyme constituting a photosynthetic pathway is PPDK.

In one preferred embodiment of the present invention for creation of cold-tolerant C4 plants, a PPDK gene that is modified into a cold-tolerant type and capable of high-level expression in target plants is used as a C4 plant genomic gene encoding an enzyme constituting a photosynthetic pathway.

Such a modification into a cold-tolerant type is accomplished, for example, by establishing “equivalence” between the amino acid sequence of PPDK to be expressed and the corresponding PPDK amino acid sequence found in plants expressing cold-tolerant PPDK (preferably at a high level). Specifically, this modification is accomplished, e.g., by introducing a mutation(s) such that the amino acid sequence is equivalent to F. brownii PPDK. More specifically, it is accomplished, for example, by introducing 17 point mutations into or downstream of Exon 15 of the maize PPDK genome-derived gene such that an amino acid sequence covering an approximately ⅙ region from the C-terminal of the maize PPDK (said amino acid sequence corresponding to the sequence downstream of No. 7682 in SEQ ID NO: 15) is identical with an amino acid sequence covering an approximately ⅙ region from the C-terminal of F. brownii PPDK, which is important for cold tolerance (said amino acid sequence corresponding to the sequence downstream of No. 7682 in SEQ ID NO: 16). For this purpose, it is desirable to use codons that occur frequently in maize. Introduction of point mutations may be carried out in a general manner well known to those skilled in the art, e.g., by PCR using a primer set(s) carrying mutations.

When used herein for amino acid sequences, the term “equivalent” or “equivalence” encompasses both the meanings that an amino acid sequence is “identical” with a target sequence and that an amino acid sequence is modified to include substitution of amino acids by other qualitatively similar amino acids, with consideration given to the nature of amino acids. Likewise, for the case where the amino acid sequence of a certain enzyme is “equivalent to the amino acid sequence of the corresponding enzyme in plants with desired characteristics,” the meaning is that the former amino acid sequence is exactly identical with the latter amino acid sequence, and that the former amino acid sequence is not exactly identical with the latter amino acid sequence, but is modified to include substitution of one or more (preferably about 1 to 40, more preferably about 1 to 30, most preferably about 1 to 20) amino acids different from those of the latter amino acid sequence so as to establish equivalence.

To obtain a PPDK gene modified into a cold-tolerant type and capable of high-level expression in target plants, for example, the above-mentioned mutations are allowed to occur exclusively in exon segments, with as many intron segments of the C4 plant genome-derived PPDK gene as possible being retained intact.

As a mutated “C4 plant genomic gene encoding an enzyme constituting a photosynthetic pathway,” a gene consisting of any one of the following DNA molecules may be used. Such a gene is particularly preferred to obtain maize plants modified to enhance their photosynthetic rate under low temperature conditions:

(a) a nucleotide sequence derived from the region downstream of Exon 15 of the maize PPDK genomic gene, whose amino acid sequence is mutated to be equivalent to the F. brownii PPDK amino acid sequence, i.e., a DNA molecule consisting of the nucleotide sequence of Nos. 1732-8508 of SEQ ID NO: 16;

(b) a DNA molecule consisting of a nucleotide sequence derived from the nucleotide sequence of Nos. 1732-8508 of SEQ ID NO: 16 by deletion, substitution, addition or insertion of one or more nucleotides, and encoding a protein possessing PPDK activity;

(c) a DNA molecule being hybridizable under stringent conditions to the DNA molecule being complementary to the DNA molecule consisting of the nucleotide sequence of Nos. 1732-8508 of SEQ ID NO: 16, and encoding a protein possessing PPDK activity; and

(d) a DNA molecule consisting of a nucleotide sequence being at least 50% (preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90%, particularly preferably at least 95%, and most preferably at least 98%) homologous to the nucleotide sequence of Nos. 1732-8508 of SEQ ID NO: 16, and encoding a protein possessing PPDK activity.

Alternatively, a gene encoding any one of the following proteins may be used:

(a) F. brownii PPDK, i.e., a protein consisting of the amino acid sequence of SEQ ID NO: 18;

(b) a protein consisting of an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 18 by deletion, substitution, addition or insertion of one or more amino acids, and possessing PPDK activity;

(c) PPDK derived from a C4 plant being cold-tolerant, or PPDK derived from a C3/C4 intermediate plant being cold-tolerant (preferably Flaveria brownii); and

(d) a protein consisting of an amino acid sequence being at least 50% (preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90%, particularly preferably at least 95%, and most preferably at least 98%) homologous to the amino acid sequence of SEQ ID NO: 18, and possessing PPDK activity.

The present invention also provides an expression cassette that comprises an expression control region such as a promoter, a downstream C4 plant genomic gene (either of the genome-derived type or the modified type), under control of said region, encoding an enzyme constituting a photosynthetic pathway, and a terminator.

Any promoter may be used in the expression cassette of the present invention as long as it is operable in plants to be transformed. Examples of a promoter available for use include promoters driving high-level expression, in green organs, of photosynthesis-related genes including PPDK (Matsuoka et al., Proc Natl Acad Sci USA, 90:9586-9590 (1993)), PEPC (Yanagisawa and Izui, J Biochem, 106:982-987 (1989) and Matsuoka et al., Plant J, 6:311-319 (1994)) and Rubisco (Matsuoka et al., Plant J, 6:311-319 (1994)); cauliflower mosaic virus 35S promoter; ubiquitin promoter (Cornejo et al., Plant Mol Biol, 23:567-581 (1993)); actin promote (McElroy et al., Plant Cell, 2:163-171 (1990)); α-tubulin promoter (Carpenter et al., Plant Mol Biol, 21:937-942 (1993)); Sc promoter (Schenk et al., Plant Mol Biol, 39:1221-1230 (1999)); pea PAL promoter; Prp1 promoter (JP 10-500312 A); hsr203J promoter (Pontier et al., Plant J, 5:507-521 (1994)); EAS4 promoter (Yin et al., Plant Physiol, 115:437-451 (1997)); PR1b1 promoter (Tornero et al., Mol Plant Microbe Interact, 10:624-634 (1997)); tap1 promoter (Mohan et al., Plant Mol Biol, 22:475-490 (1993)); and AoPR1 promoter (Warner et al., Plant J, 3:191-201 (1993)).

Any terminator may be used in the expression cassette of the present invention as long as it is operable in plants to be transformed. Examples of a terminator available for use include nos terminator, CaMV 35S terminator, gene7 terminator and protease inhibitor II terminator.

Expression cassettes comprising the following DNA molecules may be presented by way of example for the expression cassette of the present invention:

(a) a DNA molecule consisting of the nucleotide sequence of SEQ ID NO: 15;

(b) a DNA molecule consisting of a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 15 by deletion, substitution, addition or insertion of one or more nucleotides, and encoding a protein possessing PPDK activity;

(c) a DNA molecule being hybridizable under stringent conditions to the DNA molecule being complementary to the DNA molecule consisting of the nucleotide sequence of SEQ ID NO: 15, and encoding a protein possessing PPDK activity; and

(d) a DNA molecule consisting of a nucleotide sequence being at least 50% (preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90%, particularly preferably at least 95%, and most preferably at least 98%) homologous to the nucleotide sequence of SEQ ID NO: 15, and encoding a protein possessing PPDK activity; or

(a) a DNA molecule consisting of the nucleotide sequence of SEQ ID NO: 16;

(b) a DNA molecule consisting of a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 16 by deletion, substitution, addition or insertion of one or more nucleotides, and encoding a protein possessing PPDK activity;

(c) a DNA molecule being hybridizable under stringent conditions to the DNA molecule complementary to the DNA molecule consisting of the nucleotide sequence of SEQ ID NO: 16, and encoding a protein possessing PPDK activity; and

(d) a DNA molecule consisting of a nucleotide sequence being at least 50% (preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90%, particularly preferably at least 95%, and most preferably at least 98%) homologous to the nucleotide sequence of SEQ ID NO: 16, and encoding a protein possessing PPDK activity.

The expression cassette of the present invention is particularly useful in creating C4 plants modified to enhance their photosynthesis rate under low temperature conditions. The present invention also provides a recombinant vector containing such an expression cassette.

A vector used for subcloning of each DNA fragment constituting the expression cassette of the present invention may be conveniently prepared by ligating a desired gene to a recombination vector (plasmid DNA) available in the art in a general manner. Specific examples of a vector available for use include pCR2.1, pBluescript, pUC18, pUC19 and pBR322, by way of example for E. coli-derived plasmids.

A plant transformation vector is useful in introducing the expression cassette of the present invention into target plants. Any plant transformation vector may be used as long as it is capable of expressing the gene of interest in plant cells and thus producing the protein of interest. Examples include pBI221, pBI121 (both available form Clontech) and vectors derived therefrom. In particular, for transformation of monocotyledonous plants, the following vectors may be exemplified: pIG121Hm, pTOK233 (both found in Hiei et al., Plant J., 6:271-282 (1994)), pSB424 (Komari et al., Plant J., 10:165-174 (1996)), pSB11, pSB21 and vectors derived therefrom.

Preferably, a plant transformation vector at least comprises a promoter, an initiation codon, a C4 plant genomic gene (either of the genome-derived type or the modified type) encoding an enzyme constituting a photosynthetic pathway, a termination codon and a terminator. It may also comprise, as appropriate, a DNA sequence encoding a signal peptide, an enhancer sequence, 5′- and 3′-untranslated regions of the desired gene, a selective marker region and the like. Examples of a marker gene include genes resistant to antibiotics such as tetracycline, ampicillin, kanamycin, neomycin, hygromycin and spectinomycin, as well as luciferase gene, β-galactosidase gene, β-glucuronidase (GUS) gene, green fluorescent protein (GFP) gene, β-lactamase gene and chloramphenicol acetyltransferase (CAT) gene.

Techniques for plant transformation have already been established, among which the Agrobacterium method can be employed. This method is well known and can be used to transform both dicotyledonous and monocotyledonous plants (WO94/00977, WO95/06722). Gene introduction may also be accomplished, e.g., by electroporation which is a standard technique for protoplasts or by using a particle gun in a general manner. When used herein for genes, the term “introduce” or “introductions” means that a gene is put into a target plant (usually, plant cells) from outside, unless otherwise specified. The introduced gene is preferably integrated into the genome of the target plant.

Transformed cells may be selected by screening using an appropriate marker as an indicator. Transformed cells may be differentiated into transgenic plants of interest using conventional techniques.

Analysis of transformants may be carried out according to various procedures well known to those skilled in the art. For example, oligonucleotide primers are synthesized based on the DNA sequence of the introduced gene and then used in PCR to analyze the chromosomal DNAs of the transgenic plants. Alternatively, the analysis may also be accomplished by determining the presence or absence of mRNA or protein expression corresponding to the introduced gene. Further, the analysis may be accomplished by testing the resulting plants for their characteristics including cold tolerance. In a case where a genomic gene for PPDK is introduced, transformants may be analyzed for the expression level of PPDK. To determine whether transformants are cold-tolerant or not, the transformants themselves or PPDK collected therefrom may be analyzed for a decrease in PPDK activity when treated at low temperature. Procedures for these analyses are well known to those skilled in the art.

The transgenic plant of the present invention allows more expression of an enzyme constituting a photosynthetic pathway, when compared with a non-transgenic plant. The expression level is preferably an effective amount to enhance photosynthesis rate in the transgenic plant. The “effective amount to enhance photosynthesis rate” means that at a certain temperature, preferably at low temperature (e.g., at around 12° C., at around 0° C.), the transgenic plant allows more expression of the enzyme constituting a photosynthetic pathway when compared with a non-transgenic plant, so that the transgenic plant has a greater photosynthesis rate and/or it grows better or produces a desired product in a higher yield.

Further, in the transgenic plant of the present invention created using a modified genomic gene, an enzyme constituting a photosynthetic pathway is more highly expressed and/or is more resistant to deactivation, when compared with a non-transgenic plant. In this case, the level of expression/deactivation is preferably an effective amount/level to enhance photosynthesis rate in the transgenic plant. The “effective amount to enhance photosynthesis rate” is as stated previously. The “effective level to enhance photosynthesis rate” used for deactivation means that at a certain temperature, preferably at low temperature (e.g., at around 12° C., at around 0° C.), the enzyme in the transgenic plant is more resistant to deactivation when compared with a non-transgenic plant, so that the transgenic plant has a greater photosynthesis rate and/or it grows better or produces a desired product in a higher yield.

In addition to maize shown below in the Examples, the present invention can also be applied to other C4 plants including sugarcane, green amaranth, Japanese millet, foxtail millet, sorgum, millet and proso millet.

As used herein, the term “transgenic plant” encompasses not only transgenic plants (T₀ generation) regenerated from recombinant plant cells created according to the method of the present invention, but also progeny plants (e.g., T₁ generation) obtained from such transgenic plants as long as the progeny plants retain the introduced characteristics. Also, the term “plant” as used herein encompasses plant individuals as well as seeds (including germinated seeds, immature seeds), organs or portions thereof (including leaves, roots, stems, flowers, stamens, pistils, pieces thereof), cultured plant cells, calluses and protoplasts, unless otherwise specified.

The method of the present invention achieves more expression of an enzyme constituting a photosynthetic pathway (e.g., PPDK as an important enzyme in the C4 cycle, or a modified form thereof) in C4 plants. The method of the present invention further achieves enhancement of photosynthesis rate in C4 plants, enabling the C4 plants to attain cold tolerance. This in turn achieves increased production of C4 plants having PPDK, particularly increased production of maize or the like under low temperature conditions.

To date, there were some examples where a C4 plant genomic gene was expressed in C3 plants, but there was no case where a C4 plant genomic gene was introduced into C4 plants to achieve high-level expression of the gene. Since C3 plants are essentially free from genes involved in the C4 photosynthesis (or, if any, in very low amounts), C3 plants readily show effects of the introduced gene. According to the present invention, high-level expression of a C4 plant genomic gene can be achieved in C4 plants and effects of the introduced gene can be obtained without being masked by the corresponding endogenous gene. Also, in another embodiment of the present invention, the nature of a gene may be modified using point mutagenesis procedures to give greater effects under specific conditions (e.g., low temperature conditions), unlike simply improving the expression level. The present invention enables the PPDK gene involved in the C4 photosynthesis to be expressed in C4 plants at high level, thus achieving for the first time enhancement of photosynthesis rate in C4 plants, which has not been realized.

Although, as stated above, the method of the present invention is useful for transformation of an enzyme constituting the C4 photosynthetic pathway, the same procedures may also be adapted to provide C4 plants with other desired characteristics. Namely, the present invention also provides a method for highly expressing an object protein in a plant using an expression cassette that comprises a promoter, a plant genomic gene, under control of said promoter, encoding the object protein, and a terminator. The genomic gene as used here will preferably comprise a nucleotide sequence derived from the nucleotide sequence of the plant genome-derived gene encoding the object protein, by deletion, substitution, addition or insertion of one or more nucleotides in the exon(s) of the gene, with the introns of the gene retained. Although the method of the present invention is useful for transformation of C4 plants, the same procedures may also be adapted to transformation of other plants.

EXAMPLES Example 1

(Construction of #2706)

In order to introduce the unmodified maize PPDK genomic gene into maize, a gene used for transformation (#2706) was constructed as follows.

A 4.5 kb BamHI fragment covering from the latter half of Intron 1 to the first half of Intron 6 was cleaved from the maize PPDK genome cloned in λongC (Matsuoka M, 1990) and then inserted into a BamHI site of pSB11 (Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T, Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection marker, Plant J, 10:165-175, 1996). This plasmid was digested with XbaI to remove the λ arm segment and then blunt-ended with Klenow enzyme. The resulting fragment was ligated to a similarly blunt-ended 2 kb EcoRI fragment (covering from the promoter region to the middle of Intron 1) cleaved from the PPDK genome cloned in λEMBL3 (Matsuoka M, 1990). Subsequently, a 3.3 kb BamHI fragment covering from the latter half of Intron 6 to the transcription termination region was cleaved from the λongC clone and then inserted into a BamHI site of the vector prepared above, covering from the promoter to the first half of Intron 6. Finally, the bar gene ligated to the maize ubiquitin promoter, maize introns and the nos terminator was inserted into a KpnI site (FIG. 1). The DNA sequence (promoter to terminator) of the finally constructed clone #2706 is shown in SEQ ID NO: 15.

(Creation and Evaluation of Transgenic Plants)

According to the Agrobacterium method, the gene for transformation (#2706) was introduced into a maize inbred line (A188) to create a transgenic plant. At that time, a gene resistant to the herbicide Basta was used as a selective marker. The resulting transgenic plant was inbred to produce its progeny of the next generation, which were then cultivated. Cut leaves were sampled at the young-seedling stage and put into a Basta-containing medium to classify the progeny plants between individuals homozygous or heterozygous for the introduced PPDK gene (homo or hetero) and individuals null for the gene (null) (Ming-Bo Wang, Peter M. Waterhouse, A rapid and simple method of assaying plants transformed with hygromycin or PPT resistance, Plant Molecular Biology Reporter, 15:209-215 (1997)).

Subsequently, a green leaf extract was collected from leaves of each plant and subjected to Western analysis to estimate the amount of PPDK present in each progeny plant.

Further, from knee-height stage to de-tasselling stage, the photosynthesis rate was determined using photosynthesis-measuring devices (Model LI-6400, LI-COR). Two devices were always used for determination to perform simultaneous measurement on a pair of the same transformant-derived homo or hetero individual and null individual (25 pairs). Conditions under the photosynthesis rate was determined were set as follows: light level in a chamber: constant at 1000 μmol·m⁻²·s⁻¹ (LED source of artificial light); CO₂ level in a chamber: constant at 350 μmol·CO₂mol⁻¹; humidity in a chamber: not controlled in principle (loosely controlled within a range where measurement was not affected); and air stream (flow rate) in a chamber: constant at 500 μmol·s⁻¹. The photosynthesis rate was determined at leaf surface temperatures of 30° C., 20° C., 13° C. and 8° C. For determination under low temperature conditions, the photosynthesis-measuring devices and plants (leaves only) were transferred into a refrigerating room where the determination was performed. Data analysis was made by paired t-test.

(Results)

The amount of PPDK contained per g of fresh green leaves is, on average, 2484.0 μg/gfwt in the homo or hetero group and 1179.6 μg/gfwt in the null group. There was a significant difference between them at a significance level of 1%, as analyzed by paired t-test. This indicated that the introduction of the maize PPDK genomic gene resulted in an increase in the expression level of PPDK (FIG. 2A).

This increase in the expression level of PPDK was also as much as sufficient to clarify effects of the introduced gene. At a leaf surface temperature of 8° C., the photosynthesis rate was found to be improved in the homo or hetero individuals when compared with null individuals (at a significance level of 5%) (FIG. 2B).

Example 2

Next, the inventors of the present invention attempted to modify the maize PPDK genomic gene into a cold-tolerant type. In the previous studies, the inventors of the present invention had already succeeded in artificially creating a cold-tolerant PPDK gene by forming a chimeric gene between cDNAs (WO95/15385). They in turn created a modified maize PPDK genomic gene whose sequence was partially replaced with the F. brownii PPDK cDNA such that the resulting spliced mRNA was identical with mRNA prepared from the cold-tolerant chimeric gene between cDNAs. This chimeric gene was then introduced into maize, but resulting in low level expression. This result would be brought about by elimination of introns naturally occurring in the genomic gene due to partial replacement of the genomic gene with the cDNA.

For this reason, with the aim of modifying the maize PPDK genomic gene into a cold-tolerant type while leaving its structure as intact as possible, point mutations were introduced to construct a cold-tolerant modified maize PPDK genomic gene (#2838) as follows.

(Construction of #2838)

Introduction of these mutations into the genomic clone was carried out by PCR (Ho S N, Hunt H D, Horton R M, Pullen J K, Pease L R, Site-directed mutagenesis by overlap extension using the polymerase chain reaction, Gene 77:51-59 (1989)). As a PCR polymerase, ExTaq or Pyrobest (both available from Takara Shuzo Co., Ltd.) was used in order to minimize amplification errors and each fragment was subcloned every step into the plasmid vector pCR2.1 or pUC19 to confirm that there was no error in its nucleotide sequence. The mutations were introduced such that the amino acid sequence was equivalent to F. brownii PPDK, and codons used for this purpose were selected from those which occurred frequently in the maize PPDK gene. First, an approximately ⅙ region from the C-terminal of PPDK, which was important for cold tolerance (Ohta S et al., (1996); corresponding to Exon 15 and its downstream region) was divided into 6 fragments and these separate fragments were amplified using primer sets carrying mutations: F1 & R1, F2 & R2, F3 & R3, F4 & R4, F5 & R5 and F6 & R6 (SEQ ID NOs: 1 to 6 and 8 to 13). Flanking two fragments were successively ligated together by PCR and Fragments 1 to 4 were then ligated into one fragment. Finally, all fragments were ligated together to complete a XhoI-BamHI fragment (FIG. 3).

Meanwhile, a SmaI-EcoRI fragment covering from the latter half of Exon 14 to the first half of Exon 16 of the maize PPDK genomic clone was subsloned into pUC18, followed by introduction of a XboI site into Exon 15 by PCR using the primer set M4 (SEQ ID NO: 7) and mXho (SEQ ID NO: 14). This fragment was ligated to the XboI-BamHI fragment prepared above to replace the corresponding region in the 3.3 kb BamHI fragment. This fragment was inserted into a BamHI site of the vector covering from the promoter to the first half of Intron 6. Finally, the bar gene ligated to the maize ubiquitin promoter, maize introns and the nos terminator was inserted into a KpnI site (FIG. 4). The DNA sequence (promoter to terminator) of the finally constructed clone #2838 is shown in SEQ ID NO: 16.

The amino acid sequence covering an approximately ⅙ region from the C-terminal of F. brownii PPDK, which is important for cold tolerance (said amino acid sequence corresponding to the sequence downstream of No. 7682 in SEQ ID NO: 16) is shown in FIG. 5 along with the amino acid sequence covering the same region of the maize PPDK gene (said amino acid sequence corresponding to the sequence downstream of No. 7682 in SEQ ID NO: 15).

(Creation and Evaluation of Transgenic Plants)

In the same manner as shown in Example 1, the point-mutated cold-tolerant maize PPDK genomic gene (#2838) was introduced into a maize inbred line (A188) to create a transgenic plant.

Subsequently, in the same manner as shown in Example 1, the progeny plants were classified between homo or hetero individuals and null individuals, followed by Western analysis to estimate the amount of PPDK and determination of the photosynthesis rate. As in Example 1, two devices were always used for determination of the photosynthesis rate to perform simultaneous measurement on a pair of the same transformant-derived homo or hetero individual and null individual. Data analysis was also made by paired t-test.

(Result 1)

The amount of PPDK is, on average, 2742.2 μg/gfwt in the homo or hetero group and 1471.8 μg/gfwt in the null group (FIG. 6A). This indicated that the introduction of the point-mutated cold-tolerant maize PPDK genomic gene resulted in an increase in the expression level of PPDK.

Also, the photosynthesis rate was found to be improved in the homo or hetero individuals when compared with null individuals (analysis on 15 pairs) (FIG. 6B).

(Result 2)

The number of plants used for determination of the photosynthesis rate was 15 pairs at 30° C., 19 pairs at 20° C. and 21 pairs at 8° C. The amount of PPDK is shown for the respective temperatures (FIG. 7A). The homo or hetero individuals far exceeded the null individuals (at a significance level of 1%).

Also, the photosynthesis rate was found to be improved in the homo or hetero individuals when compared with null individuals (FIG. 7B).

These results indicated that the introduction of the point-mutated cold-tolerant maize PPDK genomic gene resulted in an extremely large increase in the expression level of PPDK and a further improvement in photosynthesis rate, as compared with simple introduction of the unmodified genomic PPDK. Namely, the inventors of the present invention found that the photosynthesis rate was improved even in a temperature range where no difference was observed by simply improving the expression level.

Example 3

Further, the inventors of the present invention examined PPDK activity in the transgenic maize plants having the cold tolerance-improved genomic gene introduced thereinto. In the experiment, several maize plants with different expression levels of PPDK were used along with F. brownii and a maize inbred line (A188) as controls. A green leaf extract collected from leaves of each plant was desalted on a Sephadex G25 column and then allowed to stand at 0° C. (on ice), followed by periodical verification of PPDK activity to monitor the time course of the change in PPDK activity under low temperature conditions. PPDK activity was determined in a general manner (Jenkins C L, Hatch M D, Properties and reaction mechanism of C4 leaf pyruvate, Pi dikinase, Arch Biochem Biophys, 239:53-62, 1985).

The results are shown in FIG. 8. It could be confirmed that transformants with high expression levels of PPDK were resistant to deactivation, even on ice, as in the case of F. brownii. 

1. A method for increasing the expression level of pyruvate orthophosphate dikinase (PPDK) in a C4 plant, comprising the steps of: (i) transforming the C4 plant using an expression cassette that comprises: a promoter; an isolated DNA from a 04 plant genome encoding PPDK, operably linked to said promoter; and a terminator; wherein said DNA comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence as set forth in nucleotides 1732-8508 of SEQ ID NO:15 and encodes a protein having PPDK activity; and (ii) expressing said DNA in said C4 plant, wherein expression of said PPDK is increased in the C4 plant.
 2. The method according to claim 1, wherein said DNA consists of the nucleotide sequence as set forth in nucleotides 1732-8508 of SEQ ID NO:15.
 3. The method according to claim 1, wherein said C4 plant is maize.
 4. The method according to claim 1, wherein said DNA comprises a sequence having nucleotides 1732-8508 of SEQ ID NO:15. 